Methods and compositions to elicit multivalent immune responses against dominant and subdominant epitopes, expressed on cancer cells and tumor stroma

ABSTRACT

The present invention provides a method of treating cancer by providing to a subject in need thereof an immunogenic composition comprising a nucleic acid construct encoding a polypeptide comprising CTL epitopes PSMA 288-297  and PRAME 425-433 , or a cross-reactive analogue. In embodiments of the present invention there is provided methods and compositions for inducing, entraining, and/or amplifying the immune response to MHC class-I restricted epitopes of carcinoma antigens to generate an effective anti-cancer immune response.

The present application is a continuation application of U.S. patentapplication Ser. No. 11/454,616, filed on Jun. 16, 2006, which in turnclaims the benefit of the filing date of U.S. Provisional PatentApplication No. 60/691,579, filed on Jun. 17, 2005, the entirety of eachof which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention disclosed herein is directed to inducing an MHC class-Irestricted immune response and controlling the nature and magnitude ofthe response, thereby promoting effective immunologic intervention inpathogenic processes. The invention relates to immunogenic compositionsthat can stimulate a cellular immune response against a target cell.Disclosed herein is an immunogenic composition comprising a nucleic acidconstruct encoding the CTL epitopes PRAME₄₂₅₋₄₃₃ and PSMA₂₈₈₋₂₉₇ or across-reactive analogue of either or both of epitopes. The inventionalso provides methods of using the described immunogenic composition toelicit a balanced immune response in a subject to whom such compositionsare administered.

2. Description of the Related Art

Cancer generally develops when cells in a part of the body continue togrow and divide in an unorderly manner unlike normal cells that grow,divide, and die in an orderly fashion. Although there are many kinds ofcancer, they usually start because of out-of-control growth of abnormalcells.

Usual treatment options for cancer include surgery, radiation therapy,and chemotherapy. A fourth branch of treatment is developing, which isreferred to as immunotherapy. Immunotherapies attempt to help the immunesystem recognize cancer cells, and/or to strengthen a response againstcancer cells in order to destroy the cancer. Immunotherapies includeactive and passive immunotherapies. Active immunotherapies attempt tostimulate the body's own immune system to fight the disease. Passiveimmunotherapies generally do not rely on the body to attack the disease;instead, they use immune system components (such as antibodies) createdoutside of the patient's body.

Despite various types of cancer treatments, a continuing need exists foradditional treatment options. Manipulation of the immune system by useof an anticancer vaccine is one such approach.

To generate a vaccine or other immunogenic composition, an antigen orepitope against which an immune response can be mounted is introduced toa subject. Although neoplastic (cancer) cells are derived from andtherefore are substantially identical to normal cells on a geneticlevel, many neoplastic cells are known to present tumor-associatedantigens (TuAAs). In theory, these antigens could be used by a subject'simmune system to recognize and attack the neoplastic cells as foreign.Unfortunately, neoplastic cells generally appear to be ignored by thehost's immune system.

The immune system can be categorized into two discrete effector arms.The first is innate immunity, which involves numerous cellularcomponents and soluble factors that respond to all infectiouschallenges. The other is the adaptive immune response, which iscustomized to respond specifically to precise epitopes from infectiousagents. The adaptive immune response is further broken down into twoeffector arms known as the humoral and cellular immune systems. Thehumoral arm is centered on the production of antibodies by B-lymphocyteswhile the cellular arm involves the killer cell activity of cytotoxic Tlymphocytes.

Cytotoxic T lymphocytes (CTL) do not recognize epitopes on theinfectious agents themselves. Rather, CTL detect fragments of antigensderived from infectious agents that are displayed on the surface ofinfected cells. As a result antigens are visible to CTL only after theyhave been processed by the infected cell and thus displayed on thesurface of the cell.

The antigen processing and display system on the surface of cells hasbeen well established. CTL recognize short peptide antigens, which aredisplayed on the surface in non-covalent association with class I majorhistocompatibility complex molecules (MHC). These class I peptides arein turn derived from the degradation of cytosolic proteins.

In most instances, neoplastic processes evolve to avoid the immunedefense mechanisms by employing a range of strategies that result inimmune ignorance, tolerance or deviation. Methods that effectively breakimmune tolerance or repair immune deviation against antigens expressedon cancer cells have been described in the literature (Okano F, et al. JImmunol. 2005, March 1; 174(5):2645-52; Mocellin S, et al., Exp CellRes. 2004 Oct. 1; 299(2):267-78; Banat G A, et al., Cancer ImmunolImmunother. 2001 January; 49(11):573-86) and despite their associationwith significant levels of systemic immunity, rarely result in reductionof tumor burden. Significant limiting factors impacting this process aresub-optimal trafficking, local activation and/or activity ofanti-tumoral effector cells. In fact, it has been shown in mostinstances that the intra-tumoral presence of immune cells is a rareoccurrence—compared to that associated with inflammatory processes suchas organ rejection, infections or autoimmune syndromes.

The immune response resulting from exposure to antigens (in a naturalcontext or upon vaccination) that encompass multiple epitopes isinherently associated with a hierarchy relative to the magnitude of theimmune response against different, individual epitopes. This occurs inthe case of T cell epitopes such as MHC class I and class II restrictedepitopes, where dominance and subdominance has been well documented.Dominant epitopes are those that elicit prominent and specificexpansions of T cells; whereas subdominant epitopes elicit relativelyreduced responses characterized by a limited expansion of specific Tcells with diminished functionality.

There are multiple reasons for an immune response to focus on a subsetof epitopes within an antigen, regardless of whether the antigen isnatural or engineered. These reasons include but are not limited to thefollowing: efficacy of generation of certain peptides or polypeptideprecursors within proteasomes (for class I restricted) or endosomes (forclass II restricted); their selective transport via TAP (for class Ipeptides) and alternative mechanisms to compartments where loading ontoMHC occurs; their affinity for MHC molecules relative to chaperones orthe invariant polypeptide chain that occupies the peptide-binding cleftof nascent MHC molecules and relative to other competing peptidesresulting from processing of the same or alternative substrates; thestability of the resulting MHC-peptide complex; and the functionality ofT cell repertoire.

In addition, two or more epitopes from different antigens broughttogether on the same artificial molecule assume a dominant/subdominantrelationship due to their intrinsic properties (such as those describedabove). This limits the practical applicability of composite moleculesfor the purpose of immunotherapy, particularly when co-targeting ofcancer cells (neoplastic) and stromal elements (such as neovasculature)is pursued.

SUMMARY OF THE INVENTION

To amplify immune mediated control of tumoral processes, embodiments ofthe present invention provide immune mediated attack of neovasculature,in addition to direct attack on tumor cells, as a component of a bi- ormulti-valent vaccine strategy aimed at establishing an inflammatoryenvironment within the tumor resulting in shrinkage, stabilization ordiminution of growth rate and invasion (local or systemic). Thismethodology can be more effective in controlling tumor processes thanstrategies that target either cancer cells or the neovasculature aloneand has beneficial implications in regard to therapeutic index(efficacy/safety).

Some embodiments relate to methods and compositions that modulate theimmune responses against epitopes with different intrinsic immuneproperties (e.g. dominant versus subdominant status in a givenimmunization context), in a manner consistent with increasing therelative activity of subdominant epitopes to achieve co-induction ofbalanced immune responses against multiple epitopes. This invention isuseful when co-targeting multiple antigens such as those expressed bycancer cells and/or underlying stroma.

In some embodiments, co-targeting of tumor neovasculature and cancerouscells, or of multiple antigens on cancer cells, can be achieved byimmunotherapeutic compositions comprising expression vectors such asplasmids that elicit immunity against transformed cells, tumor cells andendothelial cells of the neovasculature. Design of plasmids in a “stringof beads” format is accomplished as disclosed in U.S. Patent PublicationApplication No. 20030228634, entitled “EXPRESSION VECTORS ENCODINGEPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN” andherein incorporated by reference in its entirety. A preferred embodimentis a bivalent plasmid comprising immunogenic elements derived frommolecule(s) expressed on cancer cells and molecule(s) expressed onneovasculature. In particular embodiments of the invention, suchmolecules correspond to the PRAME and PSMA epitopes and cross-reactiveanalogues thereof.

In another embodiment, vectors such as plasmids express immunogenicelements derived from molecules co-expressed by cancer cells andneovasculature. In yet another embodiment, vectors such as plasmidsexpress immunogenic elements derived from a receptor and its ligand,where either the receptor or ligand is expressed by the neovasculature,and cancer cells express the other.

In still another embodiment, vectors encode immunogenic components frommolecules expressed by cancer cells or neovasculature (or other stromalcells) along with biological response modifiers, including modifiersthat act via antigen receptors on B and T cells and those that do not.

In some embodiments, vectors can be administered in a chronologicalsequence with other immunogenic agents—such as peptides—for the purposeof amplifying or modulating the therapeutic activity against cancercells, neovasculature or both (disclosed in U.S. Patent ApplicationPublication No. 20050079152 entitled “METHODS TO CONTROL MHC CLASSI-RESTRICTED IMMUNE RESPONSE”; U.S. Provisional Patent Application No.60/640,402, filed Dec. 29, 2004, and U.S. Publication No. 20060165711,all entitled METHODS TO ELICIT, ENHANCE, AND SUSTAIN IMMUNE RESPONSEAGAINST MHC CLASS I-RESTRICTED EPITOPES, FOR PROPHYLACTIC OR THERAPEUTICPURPOSES; and U.S. Provisional Application No. 60/691,581 filed on Jun.17, 2004, entitled MULTIVALENT IMMUNOTHERAPEUTICS FOR CARCINOMA, andU.S. patent application Ser. No. 11/455,279, entitled MUTLIVALENTIMMUNOTHERAPIES FOR CARCINOMA, filed on date even with this applicationboth entitled, each of which is herein incorporated by reference in itsentirety) and balancing the response against subdominant and dominantepitopes.

Inducing immune responses to epitopes that are “subdominant” in contextof a native antigen provides benefit in treating cancer since suchepitopes can be involved in negative selection (central or peripheral)occurring in diseased individuals. Thus, constructs encompassingmultiple copies of a subdominant epitope can be used to induce anincreased response against such an epitope while preserving immunityagainst dominant ones.

In addition, effective co-induction of immune responses against epitopesfrom different antigens presented by the same molecule can offer a morepractical approach to generate immunity against multiple antigens. Thishas direct implications for treatment and prevention of tumoral andinfectious diseases.

Overall, broader immune responses achieved by such methods andcompositions are more effective in dealing with pathogenic processes asopposed to immune responses heavily dominated by a limited number ofspecificities. In addition, practicality of multivalent vectors in suchmethods and compositions can alleviate the need to use numerouscomponents and cumbersome administration protocols to achieve balanced,multivalent responses.

Some embodiments relate to bivalent plasmids expressing PRAME and PSMAepitope sequences (such as those disclosed in the U.S. PatentApplication Publication Nos. 20030220239, 20050221440, 20050142144 andPCT Patent Publication No. PCT/US/11101 entitled “EPITOPE SEQUENCES”herein each incorporated by reference in its entirety) and methods ofuse of these compositions, individually or in combination with otherplasmids, to elicit a balanced immune response. Such methods can includean initiating or entraining step wherein the composition can bedelivered to various locations on the animal, but preferably isdelivered to the lymphatic system, for example a lymph node. Theentrainment step can include one or more deliveries of that composition,for example, spread out over a period of time or in a continuous fashionover a period of time.

The methods can further include an amplification step comprisingadministering a composition comprising a peptide immunogen, havingsubstantial similarity or functional similarity to the correspondingepitopes encoded by the nucleic acid composition. For example, theimmunogen can be a cross reactive sequence of the corresponding epitope.The amplification step can be performed one or more times, for example,at intervals over a period of time, in one bolus, or continuously over aperiod of time. Although not required in all embodiments, someembodiments can include the use of compositions that include animmunopotentiator or adjuvant.

It has been observed that by using this type of immunization protocolthat not only can the plasmid initiate an immune response, it biases theresponse and its subsequent amplification toward an effector as opposedto a regulatory character. Without this prior nucleic acid-basedimmunization, the repeated administration of peptide leads to a responseever more dominated by regulatory T cells. The long-lived bias toward aneffector response is termed entrainment.

Further embodiments include those in which the disclosed plasmids areused individually or in any combination. The peptide compositionscorresponding to these epitopes and used in the amplification portion ofthe immunization strategy can be native sequences or peptide analogssubstantially similar or functionally similar to the native epitopesequence. The peptides can be incorporated into the amplificationprotocol individually or in combinations of 2, 3, or 4 of theimmunogens. Reasons for using less than all peptide epitopes include butare not limited to the following: 1) sub-optimal expression of any ofthe antigens; 2) the patient does not express, or no longer expressesthe corresponding antigen; 3) a less robust response is being generatedto one or another of the epitopes, in which case such peptide(s) can begiven in the absence of the others in order to obtain a more balancedresponse; and 4) a peptide can be discontinued if it is generating somesort of immunotoxicity.

Additional embodiments relate to methods of modulating the immuneresponse by changing the relative number of immunogen epitopes within anucleic acid composition. These embodiments can also encompass changingthe intrinsic immunogenicity of the immunogen, for example, by encodingamino acid substitutions within the immunogen epitope.

Embodiments additionally can encompass methods of modulating the immuneresponse by selective up-regulation by peptide boost. The peptidecompositions corresponding to this amplification step can be nativesequences or peptide analogs substantially similar or functionallysimilar to the native epitope sequence. The selective up-regulation canbe achieved by administration of the peptide corresponding to thesubdominant epitope in order to obtain a balanced immune response.

Still other embodiments include plasmids that encode an analogue ofeither the PSMA or PRAME epitopes. Further embodiments can includedifferent epitopes (such as those disclosed in U.S. Patent ApplicationPublication Nos. 20030220239 and 20040180354 both entitled “EPITOPESEQUENCES” and herein incorporated by reference in their entirety) andanalogues substituted in similar combination as the epitopes expressedin the RP8 and RP12 plasmids and corresponding peptide immunogens (suchas those disclosed in U.S. Provisional Patent Application No. 60/691,889entitled “EPITOPE ANALOGUES”, filed on Jun. 17, 2005, and hereinincorporated by reference in its entirety) administered as theamplification portion of the immunization strategy.

Some embodiments relate to nucleic acid constructs encoding apolypeptide that includes one or more copies of CTL epitope PSMA₂₈₈₋₂₉₇(SEQ ID NO:6) and one or more copies of CTL epitope PRAME₄₂₅₋₄₃₃ (SEQ IDNO:5), or a cross-reactive analogue comprising 1-3 substitutions of oneor both of the epitopes, wherein the polypeptide does not include awhole antigen. The one or both epitopes can be encoded within aliberation sequence, for example. The polypeptide further can include asequence encoding one or more epitope clusters. The nucleic acidconstruct can include a PRAME epitope cluster, for example, amino acid422-509 of PRAME (SEQ ID NO:21). The nucleic acid construct can includea PSMA epitope cluster, for example, one or more epitope clusters can beamino acids 3-45 (SEQ ID NO:22) or 217-297 of PSMA (SEQ ID NO:23). ThePSMA epitope analogue can contain a I297V substitution, for example. Thenucleic acid construct further can include one or more of a nuclearimport sequence, a promoter (for example, a cytomegalovirus (CMV)promoter), a poly-A sequence, or one or more of a CpG immunostimulatorymotifs. The liberation sequence of both the PRAME and PSMA epitopes canbe located, for example, in the N-terminal portion of the encodedpolypeptide. The encoded polypeptide can be, for example, SEQ ID NO:2.The liberation sequence of both the PRAME and PSMA epitopes can belocated, for example, in the C-terminal portion of the encodedpolypeptide. For example, the encoded polypeptide can be SEQ ID NO:4.

Some embodiments relate to immunogenic compositions that include anucleic acid construct described above and elsewhere herein.

Some embodiments relate to methods of treating an individual havingcancer, the methods can include the step of administering atherapeutically effective amount of a nucleic acid construct describedabove and elsewhere herein. The nucleic acid construct can beadministered intranodally, for example. The individual can have a cancerthat expresses PRAME (SEQ ID NO:20), PSMA (SEQ ID NO:19), or both inneoplastic cells or tumor-associated neovasculature cells.

Some embodiments relate to methods of treating an individual havingcancer. The methods can include the steps of administering an effectiveamount of the nucleic acid construct described above and elsewhereherein to induce an immune response; and amplifying the immune responseby boosting with at least one peptide analogue corresponding to anepitope encoded by the nucleic acid construct. The individual can have,for example, a cancer that expresses PRAME on cancer cells and PSMA ontumor-associated vasculature cells. The individual can have a cancerthat expresses PRAME, PSMA, or both in neoplastic cells ortumor-associated neovasculature cells.

Some embodiments relate to use of an immunogenic composition or nucleicacid construct as described above and elsewhere herein in thepreparation of a medicament for the treatment of an individual havingcancer. The medicament can be for the treatment of an individual havingcancer by administering the medicament intranodally. The individual canhave a cancer that expresses PRAME, PSMA, or both in neoplastic cells ortumor-associated neovasculature cells.

Some embodiments relate to the use of an immunogenic composition or anucleic acid construct as described above and elsewhere herein in thepreparation of a medicament for use in the inducing an immune responsetargeting of tumor associated neovasculature. For example, thetumor-associated vasculature cells displays PRAME.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of two monovalent plasmids and one bivalent plasmid.

FIG. 2. ⁵¹Cr-release assay depicting the % specific cell lysis in cellsexpressing the P2, R2, and RP5 plasmids. Data are presented as follows:the x-axis shows target cells used with different effector to targetratio; the y-axis shows the corresponding percentage specific lysis.

FIG. 3. Structure of additional plasmids designed expressing both PRAMEand PSMA epitopes. Structure of the monovalent PRAME plasmid R2 isdepicted. P2 represents the monovalent PSMA plasmid.

FIG. 4. ELISpot analysis of PRAME and PSMA depicting induction ofbivalent responses achieved by plasmids encompassing epitopes from thedifferent antigens depicted in FIG. 3. Animals were immunized with 2injections of PRAME/PSMA bivalent plasmid (1 mg/ml) in bilateral lymphnodes. 5×10⁵ isolated splenocytes were incubated with 10 μg PSMA₂₈₈₋₂₉₇(SEQ ID NO:6): natural peptide or 10 μg PRAME₄₂₅₋₄₃₃ (SEQ ID NO:5)natural peptide for 42 hours prior to development. Graphs representaverage +/−SEM.

FIG. 5. Tetramer analysis of PRAME and PSMA following RP12 bivalentplasmid immunization. The data shows a bivalent immune response to PRAMEand PSMA with relative dominance of the response against the PRAMEepitope in plasmid-only primed mice.

FIGS. 6-6B. Tetramer analysis of PRAME and PSMA in mice receiving RP12or RP8 bivalent plasmid immunization followed by a PSMA₂₈₈₋₂₉₇ (I297V)(SEQ ID NO:7) peptide analogue boost (FIG. 6). Tetramer analysis ofPRAME and PSMA in individual animals following RP12 or RP8 bivalentplasmid immunization and PSMA₂₈₈₋₂₉₇ (I297V) (SEQ ID NO:7) peptideanalogue boost (FIG. 6B).

FIG. 7. ELISpot analysis in animals primed with RP12 or RP8 and boostedwith PSMA₂₈₈₋₂₉₇ (I297V) (SEQ ID NO:7) and PRAME₄₂₅₋₄₃₃ (L426Nva,L433Nle) (SEQ ID NO:30) peptide analogues.

FIG. 8. Polypeptide sequence for RP8 (SEQ ID NO:2).

FIG. 9. Polypeptide sequence for RP12 (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments relate to compositions that can elicit multivalent immuneresponses against dominant and subdominant epitopes. Some embodimentsalso relate to methods of designing the composition by: selectingantigens that are expressed by cancer cells and/or stromal(neovasculature) cells; defining epitopes that can have differentintrinsic immune properties, that constitute valid immune targets onsuch cancer or stromal cells; and modulating the relative number ofdominant and subdominant epitopes within a certain molecule (such as atherapeutic vector) by decreasing the ratio between the number ofdominant and subdominant epitopes, while providing optimal flankingresidues for appropriate generation within processing compartments.

Additional methods are described such as replacing one or multiplecopies of subdominant epitopes with analogue sequences or preferentiallypositioning epitopes within the molecule to modify the relativeimmunogenicity of such epitopes and ensure a more balanced, multivalentresponse. Testing for efficacy can follow the design of a set ofcandidate epitopes. Use of such molecules can be complemented byselective amplification of responses against subdominant epitopes, inthe context of prime-boost immunization strategies.

The general method for vaccine design can involve utilizing a definedalgorithm that starts with a natural or artificial sequence to find thecorrect ratio of dominant and subdominant epitopes for plasmids,vectors, and molecules encompassing multiple copies of dominant andsubdominant epitopes; engineering a set of compounds; in vitro and invivo characterization steps; and selection of appropriate plasmids orother vectors eliciting the desired balanced immune response.

An epitope as referred to herein, is a molecule or substance capable ofstimulating an immune response. In preferred embodiments, epitopesaccording to this definition include but are not necessarily limited toa polypeptide and a nucleic acid encoding a polypeptide, wherein thepolypeptide is capable of stimulating an immune response. In otherpreferred embodiments, epitopes according to this definition include butare not necessarily limited to peptides presented on the surface ofcells, the peptides being non-covalently bound to the binding cleft ofclass I MHC, such that they can interact with T cell receptors.

An MHC epitope as referred to herein is a polypeptide having a known orpredicted binding affinity for a mammalian class I or class II majorhistocompatibility complex (MHC) molecule.

An immune epitope referred to herein, is a polypeptide fragment that isan MHC epitope, and that is displayed on a cell in which immuneproteasomes are predominantly active. In another preferred embodiment,an immune epitope is defined as a polypeptide containing an immuneepitope according to the foregoing definition, which is flanked by oneto several additional amino acids. In another preferred embodiment, animmune epitope is defined as a polypeptide including an epitope clustersequence, having at least two polypeptide sequences having a known orpredicted affinity for a class I MHC. In yet another preferredembodiment, an immune epitope is defined as a nucleic acid that encodesan immune epitope according to any of the foregoing definitions.

SUBSTANTIAL SIMILARITY—this term is used to refer to sequences thatdiffer from a reference sequence in an inconsequential way as judged byexamination of the sequence. Nucleic acid sequences encoding the sameamino acid sequence are substantially similar despite differences indegenerate positions or modest differences in length or composition ofany non-coding regions. Amino acid sequences differing only byconservative substitution or minor length variations are substantiallysimilar. Additionally, amino acid sequences comprising housekeepingepitopes that differ in the number of N-terminal flanking residues, orimmune epitopes and epitope clusters that differ in the number offlanking residues at either terminus, are substantially similar. Nucleicacids that encode substantially similar amino acid sequences arethemselves also substantially similar.

FUNCTIONAL SIMILARITY—this term is used to refer to sequences thatdiffer from a reference sequence in an inconsequential way as judged byexamination of a biological or biochemical property, although thesequences may not be substantially similar. For example, two nucleicacids can be useful as hybridization probes for the same sequence butencode differing amino acid sequences. Two peptides that inducecross-reactive CTL responses are functionally similar even if theydiffer by non-conservative amino acid substitutions (and thus do notmeet the substantial similarity definition). Pairs of antibodies, orTCRs, that recognize the same epitope can be functionally similar toeach other despite whatever structural differences exist. In testing forfunctional similarity of immunogenicity one would generally immunizewith the “altered” antigen and test the ability of the elicited response(Ab, CTL, cytokine production, etc.) to recognize the target antigen.Accordingly, two sequences may be designed to differ in certain respectswhile retaining the same function. Such designed sequence variants areamong the embodiments of the present invention.

I. PLASMID CONSTRUCTION

Some embodiments of the present invention provide a number of plasmids,e.g., pRP8 (SEQ ID NO:1), pRP9, pRP10, pRP11, pRP12 (SEQ ID NO:3), andpRP13, having the ability to elicit or promote a bivalent responseagainst the tumor associated antigens PRAME and PSMA, specificallyagainst the epitopes PRAME₄₂₅₋₄₃₃ (SEQ ID NO:5) and PSMA₂₈₈₋₂₉₇ (SEQ IDNO:6). In particular embodiments of the invention there are provided theplasmids, pRP12 and pRP8 as immunogenic compositions. The methodologyfor generating plasmid constructs of the invention are as detailed,below.

Plasmid construction in preferred embodiments can entail stepwiseligation of sets of long complementary oligonucleotides resulting in thegeneration of DNA sequence encoding epitopes arrayed as a“string-of-beads.” These DNAs bear appropriate cohesive ends forrestriction enzymes that can be used for further ligation with DNAsencoding epitope cluster regions, which are amplified by performing PCRon cloned cDNA for PSMA or PRAME as a template. The entire insert isthen ligated into the vector backbone between Afl II and EcoR Irestriction sites. The entire coding sequence is verified by DNAsequencing. PCR-based mutagenesis can be used to generate sequenceencoding analogue epitope peptide, or to adjust the copies number ofdominant/subdominant epitopes to achieve the desired ratio. Thesequences of the two plasmids, RP8 and RP12, are described in detail anddisclosed as SEQ ID NO.1 and SEQ ID NO.3. For the specific plasmidsdescribed herein, the vector backbone is a modified version of pVAX, byInvitrogen (Carlsbad, Calif.), which has been previously disclosed inU.S. Pat. No. 6,709,844 entitled Avoidance of Undesirable ReplicationIntermediates in Plasmid Propagation, and U.S. patent application Ser.No. 09/561,572 entitled Expression Vectors Encoding Epitopes ofTarget-Associated Antigens, each of which is hereby incorporated byreference in its entirety. One of skill in the art will recognize thatthe coding sequences of the present invention can be placed in anynucleic acid vector suitable for use as a vaccine without exceeding thescope of the invention. For example, the sequences encoding the othermentioned plasmids can be inserted into the same or a similar backboneas used in pRP8 and pRP12 plasmids.

pRP8 and pRP12 are recombinant DNA plasmids that encode one polypeptidewith HLA A2-restricted CTL epitopes from PSMA (288-297) (SEQ ID NO:6)and an analogue thereof) and PRAME (425-433) (SEQ ID NO:5). Bothpolypeptides also include regions comprising epitope clusters of PSMA(3-45) (SEQ ID NO:22), (217-297) (SEQ ID NO:24) and PRAME (422-509) (SEQID NO:21). Flanking the defined PSMA and PRAME epitopes are short aminoacid sequences optimal for liberation of the epitopes in question byimmunoproteasome processing. The coding sequence for the polypeptide inthe plasmid is under the control of promoter/enhancer sequence fromcytomegalovirus (CMVp), which allows efficient transcription of mRNA forthe polypeptide upon uptake by APCs. The bovine growth hormonepolyadenylation signal (BGH polyA) at the 3′ end of the encodingsequence provides a signal for polyadenylation of the messenger toincrease its stability as well as for translocation out of the nucleusinto the cytoplasm for translation. To facilitate plasmid transport intothe nucleus after uptake, a nuclear import sequence (NIS) from simianvirus 40 (SV40) has been inserted in the plasmid backbone. The plasmidcarries two copies of a CpG immunostimulatory motif, one in the NISsequence and one in the plasmid backbone. Lastly, two prokaryoticgenetic elements in the plasmid are responsible for amplification in E.coli, the kanamycin resistance gene (Kan R) and the pMB1 bacterialorigin of replication.

A. RP8 Recombinant DNA Plasmid

For RP8, the amino acid sequence of the encoded polypeptide (297 aminoacid residues in length; SEQ ID NO:2) contains two liberation sequences,a 17 amino acid substrate at its N-terminus for PRAME₄₂₅₋₄₃₃(MKRPSIKR-SLLQHLIGL; SEQ ID NO:25) and a 66 amino acid substrate at itsC-terminus for PSMA₂₈₈₋₂₉₇(RK-GLPSIPVHPI-LV-GLPSIPVHPI-KRISPEKEEQYIAKR-GLPSIPVHPI-KRPSIK-RGLPSIPVHPV;SEQ ID NO:8). The entire polypeptide sequence of the encoded immunogen(SEQ ID NO:2) is shown in FIG. 8.

The stretch of the first 8 amino acid residues is an artificial sequencethat has been shown to facilitate processing of CTL epitopes byimmunoproteasomes. The next 9 amino acids (in italics) are PRAME₄₂₅₋₄₃₃(SEQ ID NO:5), a potent HLA A2-specific CTL epitope that triggers stronganti-tumor immune responses in both in vitro immunization of human PBMCand in vivo immunization in mice. This PRAME epitope sequence isfollowed by a segment (amino acid 18-105 of the immunogen) ofPRAME₄₂₂₋₅₀₉, comprising two epitope clusters: PRAME₄₂₂₋₄₄₃ (SEQ IDNO:26) and PRAME₄₅₉₋₄₈₇ (SEQ ID NO:27). Two PSMA epitope clusters (initalics), PSMA₃₋₄₅ (SEQ ID NO:22) (amino acid 108-150;) and PSMA₂₁₇₋₂₉₇(SEQ ID NO:24) (amino acid 151-231), are placed after the PRAME epitopecluster. These and other PRAME and PSMA epitope clusters have beendisclosed in U.S. patent application Ser. Nos. 10/117,937, 11/067,064,and 11/067,159, each entitled Epitope Sequences and each of which ishereby incorporated by reference in its entirety. These epitope clusterscontain a number of predicted HLA A2-specific epitopes and thus can beuseful in generating a response to immune epitopes (described in U.S.Patent Application Publication No. 20030215425 entitled EPITOPESYNCHRONIZATION IN ANTIGEN PRESENTING CELLS and U.S Patent ApplicationPublication Nos. 20030228634; 20040132088; and 20040203051, entitledEPITOPE CLUSTERS, each of which is hereby incorporated by reference inits entirety). A “string-of-beads” epitope array with multiple copies ofPSMA₂₈₈₋₂₉₇ (GLPSIPVHPI (SEQ ID NO:6); in boldface) constitutes the restof the polypeptide (amino acid 232-297). Four copies of PSMA₂₈₈₋₂₉₇ areincorporated with the last copy being an analogue (GLPSIPVHPV; SEQ IDNO:7). Both the native PSMA₂₈₈₋₂₉₇ and its analogue have been shown toinduce significant CTL responses in both in vitro immunization of humanPBMC and in vivo immunization in mice with the analogue displayingelevated MHC class I binding and immunogenicity. Between PSMA₂₈₈₋₂₉₇epitope sequences are short amino acid sequences designated to be“cleavage helper sequences” to facilitate the processing and liberationof the epitope. These two epitopes are thus encoded in such a mannerthat they can be expressed, processed, and presented by pAPCs.

B. RP12 Recombinant DNA Plasmid

For the RP12 plasmid, the amino acid sequence of the encoded polypeptide(275 amino acid residues in length; SEQ ID NO:4) contains one amino acidsubstrate or liberation sequence and a hybrid “string-of-beads”encompassing a substrate at its C-terminus for the liberation of boththe PRAME and PSMA epitopes. The entire polypeptide sequence of theencoded immunogen is shown in FIG. 9. The liberation sequencerepresented as SEQ ID NO:9 is as follows:KR-SLLQHLIGL-GDAAY-SLLQHLIGL-ISPEKEEQYIA-SLLQHLIGL-KRPSIKR-GLPSIPVHPV.

Segments of amino acid 2-44, 45-126, and 127-213 of the encodedimmunogen are epitope clusters joined one to the next: PSMA₃₋₄₅ (SEQ IDNO:22), PSMA₂₁₇₋₂₉₇ (SEQ ID NO:23), and PRAME₄₂₂₋₅₀₉ (SEQ ID NO:21),respectively. In the “string-of-beads” hybrid substrate, there are 3copies of PRAME₄₂₅₋₄₃₃ (SLLQHLIGL; in boldface; SEQ ID NO:5) and onecopy of PSMA₂₈₈₋₂₉₇ analogue (GLPSIPVHPV; in sans serif boldface; SEQ IDNO:7) at the C-terminus of the polypeptide. Between the PRAME₄₂₅₋₄₃₃ andPSMA₂₈₈₋₂₉₇ epitope sequences are the short amino acid sequencesdesignated to be “cleavage helper sequences” to facilitate processingand liberation of the epitopes. These two epitopes are thus encoded insuch a manner that they can be expressed, processed, and presented bypAPCs.

Further details on the RP12 plasmid are disclosed in U.S. ProvisionalPatent Application No. 60/691,579, filed on Jun. 17, 2005, entitledMETHODS AND COMPOSITIONS TO ELICIT MULTIVALENT IMMUNE RESPONSES AGAINSTDOMINANT AND SUBDOMINANT EPITOPES, EXPRESSED ON CANCER CELLS AND TUMORSTROMA, incorporated herein by reference in its entirety. All otherplasmids were constructed in a similar fashion using the methodology asapplied to RP8 and RP12. The plasmid R2, also referred to as pCTLR2, isdisclosed in the Examples. The P2 plasmid as shown in FIGS. 1 and 3, isa monovalent PSMA plasmid. The RP5 plasmid encompasses elements fromboth P2 and R2.

Various methodologies for constructing or designing plasmids are wellestablished in the art as would be known to the skilled artisan. Suchmethodologies are described in many references, such as, for example,Molecular Cloning, Sambrook J and Russell D. W., CSHL Press, (2001),specifically incorporated herein by reference.

In constructing the nucleic acids encoding the polypeptide epitopes ofthe invention, the gene sequence of the associated tumor associatedantigen (e.g., PRAME and PSMA) can be used, or the polynucleotide can beassembled from any of the corresponding codons. For a 10 amino acidepitope this can constitute on the order of 10⁶ different sequences,depending on the particular amino acid composition. While large, this isa distinct and readily definable set representing a miniscule fractionof the >10¹⁸ possible polynucleotides of this length, and thus in someembodiments, equivalents of a particular sequence disclosed hereinencompass such distinct and readily definable variations on the listedsequence. In choosing a particular one of these sequences to use in avaccine, considerations such as codon usage, self-complementarity,restriction sites, chemical stability, etc. can be used as will beapparent to one skilled in the art.

An epitope cluster as contemplated in the present invention is apolypeptide, or a nucleic acid sequence encoding it, that is a segmentof a native protein sequence comprising two or more known or predictedepitopes with binding affinity for a shared MHC restriction element,wherein the density of epitopes within the cluster is greater than thedensity of all known or predicted epitopes with binding affinity for theshared MHC restriction element within the complete protein sequence.Epitope clusters and their uses are described in U.S. Patent ApplicationPublication Nos. 20030220239, 20050221440, 20050142144; 20030215425,20030228634, 20040132088, 20040203051 and PCT Patent ApplicationPublication No. PCT/US/11101; all of which are incorporated herein byreference in their entirety.

A substrate or liberation sequence as employed in the present invention,is a designed or engineered sequence comprising or encoding a PRAMEand/or PSMA epitope embedded in a larger sequence that provides acontext allowing the PRAME and/or PSMA epitope to be liberated byimmunoproteasomal processing, directly or in combination with N-terminaltrimming or other processes.

The following are additional examples of encoded polypeptide sequencesthat can be used in some embodiments, for example, they can be encodedby the various plasmids or used in the methods, etc.

R2 (SEQ ID NO: 10) MALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNKRSLLQHLIGLGDAAYSLLQHLIGLISPEKEEQYIASLLQHLIGLKRPSIKRSLL QHLIGL P2(SEQ ID NO: 11) MNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIRKGLPSIPVHPILVGLPSIPVHPIKRISPEKEEQYIAKRGLPSIPVHPIKRPSIKRGLPSIPVHPI RP5 (SEQ ID NO: 12)MISPEKEEQYIASLLQHLIGLKRSLLQHLIGLKRPSIKRSLLQHLIGLALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNKLNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIRKGLPSIPVHPILVGLPSIPVHPIKRISPEKEEQYIAKRGLPSIPVHPIKRPSIKRGLPSIPVHPI RP9 (SEQ ID NO: 14)MISPEKEEQYIASLLQHLIGLKRPSIKRSLLQHLIGLALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNKLNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIRKGLPSIPVHPILVGLPSIPVHPVKRGLPSIPVHPVK RPSVKRGLPSIPVHPV RP 10(SEQ ID NO: 15) MISPEKEEQYIASLLQHLIGLALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNKLNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIRKGLPSIPVHPILVGLPSIPVHPVKRGLPSIPVHPVKRPSVKRGLPSIPVHPV RP11 (SEQ ID NO: 16)MKRSLLQHLIGLKRPSIKRSLLQHLIGLALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNKLNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIRKGLPSIPVHPVLVGLPSIPVHPVKRISPEKEEQYIAKRGLPSIPV HPIKRPSIKRGLPSIPVHPVRP13 (SEQ ID NO: 18) MKRSLLQHLIGLKRPSIKRSLLQHLIGLALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPNKLNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPVLVGLPSIPVHPVKRISPEKEEQYIAKRGLPSIPVHPIKRPSIKRGLPSIPVH PV

II. IMMUNOGENIC COMPOSITIONS OF THE PRESENT INVENTION

The present invention contemplates the use of multiple moleculesexpressed by cancer cells and by the neovasculature as therapeutictargets in the treatment of cancer by active immunotherapy. Suchmolecules include tumor-associated antigens (TuAAs) which are antigensexpressed by the cancer cell itself or associated with non-cancerouscomponents of the tumor, such as tumor-associated neovasculature orother stroma. Determination of TuAA expression profiles can help tomatch a patient's cancer condition or type with an appropriateimmunotherapeutic agent or regimen. In particular embodiments, epitopesof the tumor associated antigens PRAME and PSMA are employed indesigning bivalent plasmids that can elicit a strong immune response ina subject to whom such plasmid are administered as a cancertherapeutics. Cross-reactive analogues of PRAME and PSMA are alsocontemplated in the embodiments of the present invention.

The tumor associated antigen PRAME (SEQ ID NO:20), employed in thepresent invention, is also known as MAPE, DAGE, and 01P4. PRAME is knownin the art as a cancer-testis (CT) antigen. However, unlike many CTantigens, such as: MAGE, GAGE and BAGE, it is expressed in acute myeloidleukemias. PRAME as a TuAA is disclosed in U.S. Pat. No. 5,830,753,incorporated herein by reference in its entirety. In preferredembodiments, the present invention provides epitopes of PRAME andanalogues thereof.

Another TuAA employed in the present invention is the prostate-specificmembrane antigen (PSMA) (SEQ ID NO:19). PSMA is found to be highlyexpressed in prostate cancer cells. However, PSMA expression is alsonoted in normal prostate epithelium and in the neovasculature ofnon-prostatic tumors. PSMA as an anti-neovasculature preparation isdisclosed in U.S. Provisional Patent Application No. 60/274,063, andU.S. Patent Publication Application Nos. 20030046714 and 20050260234;each of which is incorporated herein by reference in its entirety. PSMAas a TuAA is described in U.S. Pat. No. 5,538,866 incorporated herein byreference in its entirety. In preferred embodiments, the presentinvention provides epitopes of PSMA and analogues thereof.

Cross-reactive analogue as used herein may refer to a peptide comprising1-3 amino acid substitutions, and/or one amino acid deletion or additionas compared to the native peptide sequence that induces effectorfunction (e.g., cytolysis or cytokine secretion) distinguishable frombackground, from a CTL reactive with the native peptide. In preferredembodiments effector function is at least 30, 50, 60, 70, or 80% of thatinduced by the native peptide.

In some embodiments of the present invention, peptides comprising thenative sequence or analogues (cross-reactive) of PRAME and PSMA may alsobe administered as a peptide boost in combination with the plasmids ofthe invention. Native peptide sequences and peptide analogues of PRAMEand PSMA, are disclosed in U.S. Patent Application No. 20060057673 andU.S. Provisional Patent Application No. 60/691,889, each of which ishereby incorporated by reference in its entirety. The peptide analogues,PRAME₄₂₅₋₄₃₃ L426Nva, L433Nle (SEQ ID NO:30) and PSMA₂₈₈₋₂₉₇ I297V (SEQID NO:7) are described in U.S. Provisional Application No. 60/580,962;U.S. patent application Ser. No. 11/155,929; U.S. ProvisionalApplication No. 60/581,001; U.S. patent application Ser. No. 11/156,253;U.S. patent application Ser. No. 11/156,369 U.S. Provisional PatentApplication No. 60/691,889, U.S. patent application Ser. No. 11/455,278,entitled PRAME PEPTIDE ANALOGUES, U.S. patent application Ser. No.11/454,633, entitled PSMA PEPTIDE ANALOGUES, and U.S. patent applicationSer. No. 11/454,300, entitled MELANOMA ANTIGEN PETIDE ANALOGUES each ofwhich is hereby incorporated by reference in its entirety.

As discussed above, some embodiments relate to immunogenic compositionsfor the treatment of cancer comprising plasmids encoding CTL epitopes ofPRAME and PSMA and cross-reactive analogues thereof. Such an immunogeniccomposition can elicit a robust or strong cell-mediated immune responseto target a particular cancer thereby eliminating, eradicating orameliorating the cancer in a subject.

III. ENTRAINING-AND-AMPLIFYING THERAPEUTICS FOR ADMINISTRATION

In a preferred embodiment, the present invention provides an immunogeniccomposition comprising a nucleic acid construct encoding the CTLepitopes PRAME₄₂₅₋₄₃₃ (SEQ ID NO:5) and PSMA₂₈₈₋₂₉₇ (SEQ ID NO:6) or across-reactive analogue of either or both these epitopes. In someembodiments of the invention a plasmid prime/peptide boost approach maybe employed wherein the recombinant DNA plasmid expressing the PRAME andPSMA epitopes may be administered in conjunction with a syntheticpeptides such as a PRAME and or PSMA peptide or analogue thereof.

The immunogenic composition of the invention comprising a nucleic acidconstruct encoding the CTL epitopes PRAME₄₂₅₋₄₃₃ and PSMA₂₈₈₋₂₉₇ or across-reactive analogue of one or both epitopes, can be delivered vialymph node injection, directly into the organs where the immuneresponses are initiated and amplified, according to an optimizedimmunization schedule. Embodiments of the current invention can beadministered to patients with tumor tissue that express HLA-A2,particularly HLA-A*0201. Therefore, the immunogenic compositioncomprising a plasmid and one or more peptides or analogues thereof canbe administered in treating a cancer in a subject. The disclosedembodiments of the present invention relate to entrain-and-amplifytherapeutics for cancer that can be used to achieve a bi- or multivalentattack, offering the advantage of increasing the sensitivity of thetumor to attack.

Therefore, in particular embodiments, the present invention providesbivalent entraining-and-amplifying therapeutics for the treatment ofcancer. Such bivalent therapeutics can target more than one antigen on atumor cell. In instances where more than a single antigen on a tumorcell is targeted, the effective concentration of antitumor therapeuticis increased accordingly. Attack on stroma associated with the tumor,such as vasculature, can increase the accessibility of the tumor cellsto the agent(s) targeting them. Thus, even an antigen that is alsoexpressed on some normal tissue can receive greater consideration as atarget antigen if the other antigens to be targeted in a bi- ormultivalent attack are not also expressed by that tissue. The plasmidsof the current invention can be used in conjunction with additionalplasmids that express other epitopes, and corresponding amplifyingpeptides, to create therapeutic protocols of higher valency. Exemplaryimmunogenic products are disclosed in U.S. Provisional PatentApplication No. 60/691,581, filed on Jun. 17, 2005 and U.S. patentapplication Ser. No. 11/455,279, filed on date even with the instantapplication, each entitled MULTIVALENT ENTRAIN-AND-AMPLIFYIMMUNOTHERAPEUTICS FOR CARCINOMA, and each incorporated by reference inits entirety.

An “entraining” immunogen as contemplated in the present inventionincludes in many embodiments an induction that confers particularstability on the immune profile of the induced lineage of T cells.

As contemplated in the present invention, the term “amplifying oramplification”, as of a T cell response, includes in many embodiments aprocess for increasing the number of cells, the number of activatedcells, the level of activity, rate of proliferation, or similarparameter of T cells involved in a specific response.

The entrain-and-amplify protocol employed in the present invention isdescribed in greater detail in U.S. Patent Publication No. 20050079152,U.S. Provisional Patent Application No. 60/640,402, and U.S. patentapplication Ser. No. 11/323,572 each entitled “METHODS TO ELICIT,ENHANCE AND SUSTAIN IMMUNE RESPONSES AGAINST MHC CLASS I-RESTRICTEDEPITOPES, FOR PROPHYLACTIC OR THERAPEUTIC PURPOSES” which areincorporated herein by reference in their entirety.

IV. BIOLOGICAL RESPONSE MODIFIERS (BRMS) OR IMMUNOPOTENTIATORS

In some embodiments, the present invention may further employ abiological response modifier (BRM) or immunopotentiator in conjunctionwith the immunogenic compositions comprising a recombinant DNA plasmidencoding the CTL epitopes PRAME and PSMA, in eliciting an immuneresponse. The immunopotentiators or BRMs contemplated by the presentinvention can act in an immunosuppressive or immunostimulatory manner tomediate an immune response. Immunopotentiators or BRMs of the presentinvention may refer to any molecule that modulates the activity of theimmune system, or the cells thereof, through an interaction other thanwith an antigen receptor. BRMs as contemplated in the present inventionmay further include natural or synthetic small organic molecules thatexert immune modulating effects by stimulating pathways of innateimmunity.

In particular embodiments, the present invention also contemplatesimmunopotentiators or BRMS which may include, but are not limited to,for example: cytokines such as IL-12, IL-18, GM-CSF, flt3 ligand(flt3L), interferons, TNF-alpha, and the like; chemokines such as IL-8,MIP-3alpha, MIP-1alpha, MCP-1, MCP-3, RANTES, and the like. Otherexamples of BRMs that may be utilized in the present invention aremolecules that trigger cytokine or chemokine production, such as ligandsfor Toll-like receptors (TLRs), peptidoglycans, LPS or analogues,unmethylated CpG oligodeoxynucleotides (CpG ODNs); dsRNAs such asbacterial dsDNA (which contains CpG motifs) and synthetic dsRNA(polyl:C) on APC and innate immune cells that bind to TLR9 and TLR3,respectively. One class of BRM includes mostly small organic natural orsynthetic molecules, which exert immune modulating effects bystimulating pathways of innate immunity. Thus, small molecules that bindto TLRs such as a new generation of purely synthetic anti-viralimidazoquinolines, e.g., imiquimod and resiquimod, that have been foundto stimulate the cellular path of immunity by binding the TLRs 7 and 8(Hemmi, H. et al., Nat Immunol 3: 196-200, 2002; Dummer, R. et al.,Dermatology 207: 116-118, 2003; each of which is incorporated herein byreference in its entirety) may be employed. BRMs may further includeimmunopotentiating adjuvants that activate pAPC or T cells including,for example: endocytic-Pattern Recognition Receptor (PRR) ligands,quillaja saponins, tucaresol and the like.

V. METHODS OF DELIVERING COMPOSITIONS OF THE PRESENT INVENTION

In the present invention, the preferred administration of theimmunogenic composition comprising recombinant DNA plasmids encoding theCTL epitopes PRAME and PSMA, or such plasmids followed by one or morepeptide(s) as one or more boost, is via lymph node injection. Otherimmunization protocols, for example, using plasmid for other than theinitiation dose(s), relying on plasmid alone, or utilizing other typesof boosting reagents, while less preferred embodiments, are not excludedfrom the scope of the invention. Embodiments of the present inventionencompass bivalent plasmids expressing both of the immunogens PRAME andPSMA. In delivering the immunogenic compositions of the invention to asubject in need thereof, lymph node injection is preferred as it allowsfor delivery directly into the organs where the immune responses areinitiated and amplified according to an optimized immunization schedule.

To introduce the immunogenic composition into the lymphatic system ofthe patient the composition is preferably directed to a lymph vessel,lymph node, the spleen, or other appropriate portion of the lymphaticsystem. In some embodiments each component is administered as a bolus.In other embodiments, one or more components are delivered by infusion,generally over several hours to several days. Preferably, thecomposition is directed to a lymph node such as an inguinal or axillarynode by inserting a catheter or needle to the node and maintaining thecatheter or needle throughout the delivery. Suitable needles orcatheters are available made of metal or plastic (e.g., polyurethane,polyvinyl chloride (PVC), TEFLON, polyethylene, and the like). Ininserting the catheter or needle into the inguinal node for example, theinguinal node is punctured under ultrasonographic control using aVialon™ Insyte W™ cannula and catheter of 24G3/4 (Becton Dickinson, USA)which is fixed using Tegaderm™ transparent dressing (Tegaderm™, St.Paul, Minn., USA). An experienced radiologist generally does thisprocedure. The location of the catheter tip inside the inguinal lymphnode is confirmed by injection of a minimal volume of saline, whichimmediately and visibly increases the size of the lymph node. The latterprocedure allows confirmation that the tip is inside the node. Thisprocedure can be performed to ensure that the tip does not slip out ofthe lymph node and can be repeated on various days after implantation ofthe catheter.

The therapeutic composition(s) of the present invention may beadministered to a patient in a manner consistent with standard vaccinedelivery protocols that are well known to one of ordinary skill in theart. Methods of administering immunogenic compositions of the presentinvention comprising plasmids and peptides or peptide analogues of theTuAAs PRAME and PSMA include, without limitation, transdermal,intranodal, perinodal, oral, intravenous, intradermal, intramuscular,intraperitoneal, and mucosal administration, delivery by injection orinstillation or inhalation. A particularly useful method of vaccinedelivery to elicit a CTL response is disclosed in Australian Patent No.739189; U.S. Pat. Nos. 6,994,851 and 6,977,074 both entitled “A METHODOF INDUCING A CTL RESPONSE”.

Various parameters can be taken into account in delivering oradministering an immunogenic composition to a subject. In addition, adosage regimen and immunization schedule may be employed. Generally theamount of the components in the therapeutic composition will vary frompatient to patient and from antigen to antigen, depending on suchfactors as: the activity of the antigen in inducing a response; the flowrate of the lymph through the patient's system; the weight and age ofthe subject; the type of disease and/or condition being treated; theseverity of the disease or condition; previous or concurrent therapeuticinterventions; the capacity of the individual's immune system tosynthesize antibodies; the degree of protection desired; the manner ofadministration and the like, all of which can be readily determined bythe practitioner.

In general the therapeutic composition may be delivered at a rate offrom about 1 to about 500 microliters/hour or about 24 to about 12000microliters/day. The concentration of the antigen is such that about 0.1micrograms to about 10,000 micrograms of the antigen will be deliveredduring 24 hours. The flow rate is based on the knowledge that eachminute approximately about 100 to about 1000 microliters of lymph fluidflows through an adult inguinal lymph node. The objective is to maximizelocal concentration of vaccine formulation in the lymph system. Acertain amount of empirical investigation on patients will be necessaryto determine the most efficacious level of infusion for a given vaccinepreparation in humans.

In particular embodiments, the immunogenic composition of the presentinvention may be administered as a number of sequential doses. Suchdoses may be 2, 3, 4, or more doses as is needed to obtain theappropriate immune response. In further embodiments of the presentinvention, it is contemplated that the doses of the immunogeniccomposition would be administered within about seconds or minutes ofeach other into the right or left inguinal lymph nodes. For example, theplasmid (prime) may first be injected into the right lymph node followedwithin seconds or minutes by a second plasmid into the right or leftinguinal lymph nodes. In other instances the combination of one or moreplasmid expressing one or more immunogens may be administered. It ispreferred that the subsequent injection following the first injectioninto the lymph node be within at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore minutes but not greater than about 30, 40, 50, or 60 minutes of thefirst injection. Similar considerations apply to the administration oftwo peptides individually to the right and left lymph nodes. It may bedesirable to administer the doses of the immunogenic composition of theinvention at an interval of days, where several days (1, 2, 3, 4, 5, 6,or 7, or more days) lapse between subsequent administrations. In otherinstances it may be desirable for subsequent administration(s) of thecompositions of the invention to be administered via bilateral inguinallymph node injection within about 1, 2, 3, or more weeks or within about1, 2, 3, or more months following the initial dose administration.

Administration may be in any manner compatible with the dosageformulation and in such amount as will be therapeutically effective. Aneffective amount or dose of an immunogenic composition of the presentinvention is that amount needed to provide a desired response in thesubject to be treated.

In addition to those already disclosed in this application, thefollowing applications are hereby expressly incorporated by reference intheir entireties. Useful methods for using the disclosed analogs ininducing, entraining, maintaining, modulating and amplifying class IMHC-restricted T cell responses, and particularly effector and memoryCTL responses to antigen, are described in U.S. Pat. Nos. 6,994,851(Feb. 7, 2006) and 6,977,074 (Dec. 20, 2005) both entitled “A Method ofInducing a CTL Response”; U.S. Provisional Application No. 60/479,393,filed on Jun. 17, 2003, entitled “METHODS TO CONTROL MHC CLASSI-RESTRICTED IMMUNE RESPONSE”; and U.S. patent application Ser. No.10/871,707 (Pub. No. 2005 0079152) and Provisional U.S. PatentApplication No. 60/640,402 filed on Dec. 29, 2004, both entitled“Methods to elicit, enhance and sustain immune responses against MHCclass I-restricted epitopes, for prophylactic or therapeutic purpose”.The analogs can also be used in research to obtain further optimizedanalogs. Numerous housekeeping epitopes are provided in U.S. applicationSer. Nos. 10/117,937, filed on Apr. 4, 2002 (Pub. No. 20030220239 A1),and 10/657,022 (20040180354), and in PCT Application No.PCT/US2003/027706 (Pub. No. WO04022709A2), filed on Sep. 5, 2003; andU.S. Provisional Application Nos. 60/282,211, filed on Apr. 6, 2001;60/337,017, filed on Nov. 7, 2001; 60/363,210 filed on Mar. 7, 2002; and60/409,123, filed on Sep. 5, 2002; each of which applications isentitled “Epitope Sequences”. The analogs can further be used in any ofthe various modes described in those applications. Epitope clusters,which may comprise or include the instant analogs, are disclosed andmore fully defined in U.S. patent application Ser. No. 09/561,571, filedon Apr. 28, 2000, entitled EPITOPE CLUSTERS. Methodology for using anddelivering the instant analogs is described in U.S. patent applicationSer. Nos. 09/380,534 and 6977074 (Issued Dec. 20, 2005) and in PCTApplication No. PCTUS98/14289 (Pub. No. WO9902183A2), each entitled A“METHOD OF INDUCING A CTL RESPONSE”. Beneficial epitope selectionprinciples for such immunotherapeutics are disclosed in U.S. patentapplication Ser. No. 09/560,465, filed on Apr. 28, 2000, Ser. No.10/026,066 (Pub. No. 20030215425 A1), filed on Dec. 7, 2001, and Ser.No. 10/005,905 filed on Nov. 7, 2001, all entitled “EpitopeSynchronization in Antigen Presenting Cells”; U.S. Pat. No. 6,861,234(issued 1 Mar. 2005; application Ser. No. 09/561,074), entitled “Methodof Epitope Discovery”; Ser. No. 09/561,571, filed Apr. 28, 2000,entitled EPITOPE CLUSTERS; Ser. No. 10/094,699 (Pub. No. 20030046714A1), filed Mar. 7, 2002, entitled “Anti-Neovasculature Preparations forCancer”; application Ser. Nos. 10/117,937 (Pub. No. 20030220239 A1) andPCTUS02/11101 (Pub. No. WO02081646A2), both filed on Apr. 4, 2002, andboth entitled “EPITOPE SEQUENCES”; and application Ser. Nos. 10/657,022and PCT Application No. PCT/US2003/027706 (Pub. No. WO04022709A2), bothfiled on Sep. 5, 2003, and both entitled “EPITOPE SEQUENCES”. Aspects ofthe overall design of vaccine plasmids are disclosed in U.S. patentapplication Ser. No. 09/561,572, filed on Apr. 28, 2000, entitled“Expression Vectors Encoding Epitopes of Target-Associated Antigens” and10/292,413 (Pub. No. 20030228634 A1), filed on Nov. 7, 2002, entitled“Expression Vectors Encoding Epitopes of Target-Associated Antigens andMethods for their Design”; 10/225,568 (Pub No. 2003-0138808), filed onAug. 20, 2002, PCT Application No. PCT/US2003/026231 (Pub. No. WO2004/018666), filed on Aug. 19, 2003, both entitled “EXPRESSION VECTORSENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS”; and U.S. Pat. No.6,709,844, entitled “AVOIDANCE OF UNDESIRABLE REPLICATION INTERMEDIATESIN PLASMID PROPAGATION”. Specific antigenic combinations of particularbenefit in directing an immune response against particular cancers aredisclosed in Provisional U.S. patent Application No. 60/479,554, filedon Jun. 17, 2003 and U.S. patent application Ser. No. 10/871,708, filedon Jun. 17, 2004 and PCT Patent Application No. PCT/US2004/019571 (Pub.No. WO 2004/112825), all entitled “Combinations of tumor-associatedantigens in vaccines for various types of cancers”. Antigens associatedwith tumor neovasculature (e.g., PSMA, VEGFR2, Tie-2) are also useful inconnection with cancerous diseases, as is disclosed in U.S. patentapplication Ser. No. 10/094,699 (Pub. No. 20030046714 A1), filed Mar. 7,2002, entitled “Anti-Neovasculature Preparations for Cancer”. Methods totrigger, maintain, and manipulate immune responses by targetedadministration of biological response modifiers are disclosed U.S.Provisional Application No. 60/640,727, filed on Dec. 29, 2004. Methodsto bypass CD4+ cells in the induction of an immune response aredisclosed in U.S. Provisional Application No. 60/640,821, filed on Dec.29, 2004. Exemplary diseases, organisms and antigens and epitopesassociated with target organisms, cells and diseases are described inU.S. Application No. 6977074 (issued Dec. 20, 2005) filed Feb. 2, 2001and entitled “METHOD OF INDUCING A CTL RESPONSE”. Exemplary methodologyis found in U.S. Provisional Application No. 60/580,969, filed on Jun.17, 2004, and U.S. Patent Application No. 2006-0008468-A1, published onJan. 12, 2006, both entitled “COMBINATIONS OF TUMOR-ASSOCIATED ANTIGENSIN DIAGNOTISTICS FOR VARIOUS TYPES OF CANCERS”. Methodology andcompositions are also disclosed in U.S. Provisional Application No.60/640,598, filed on Dec. 29, 2004, entitled “COMBINATIONS OFTUMOR-ASSOCIATED ANTIGENS IN COMPOSITIONS FOR VARIOUS TYPES OF CANCER”.The integration of diagnostic techniques to assess and monitor immuneresponsiveness with methods of immunization including utilizing theinstant analogs is discussed more fully in Provisional U.S. PatentApplication No. 60/580,964 filed on Jun. 17, 2004 and U.S. PatentApplication No. US-2005-0287068-A1, published on Dec. 29, 2005) bothentitled “Improved efficacy of active immunotherapy by integratingdiagnostic with therapeutic methods”. The immunogenic polypeptideencoding vectors are disclosed in U.S. patent application Ser. No.10/292,413 (Pub. No. 20030228634 A1), filed on Nov. 7, 2002, entitledExpression Vectors Encoding Epitopes of Target-Associated Antigens andMethods for their Design, and in U.S. Provisional Application No.60/691,579, filed on Jun. 17, 2005, entitled “Methods and compositionsto elicit multivalent immune responses against dominant and subdominantepitopes, expressed on cancer cells and tumor stroma”. Additional usefuldisclosure, including methods and compositions of matter, is found inU.S. Provisional Application No. 60/691,581, filed on Jun. 17, 2005,entitled “Multivalent Entrain-and-Amplify Immunotherapeutics forCarcinoma”. Further methodology, compositions, peptides, and peptideanalogs are disclosed in U.S. Provisional Application Nos. 60/581,001and 60/580,962, both filed on Jun. 17, 2004, and respectively entitled“SSX-2 PEPTIDE ANALOGS” and “NY-ESO PEPTIDE ANALOGS.” Each of theapplications and patents mentioned in the above paragraphs is herebyincorporated by reference in its entirety for all that it teaches.Additional analogs, peptides and methods are disclosed in U.S. PatentApplication Publication No 20060063913, entitled “SSX-2 PEPTIDEANALOGS”; and U.S. Patent Publication No. 2006-0057673 A1, published onMar. 16, 2006, entitled “EPITOPE ANALOGS”; and PCT ApplicationPublication No. WO/2006/009920, entitled “EPITOPE ANALOGS”; all filed onJun. 17, 2005. Further methodology and compositions are disclosed inU.S. Provisional Application No. 60/581,001, filed on Jun. 17, 2004,entitled “SSX-2 PEPTIDE ANALOGS”, and to U.S. Provisional ApplicationNo. 60/580,962, filed on Jun. 17, 2004, entitled “NY-ESO PEPTIDEANALOGS”; each of which is incorporated herein by reference in itsentirety. As an example, without being limited thereto each reference isincorporated by reference for what it teaches about class IMHC-restricted epitopes, analogs, the design of analogs, uses ofepitopes and analogs, methods of using and making epitopes, and thedesign and use of nucleic acid vectors for their expression. Otherapplications that are expressly incorporated herein by reference are:U.S. patent application Ser. No. 11/156,253 (Publication No.20060063913), filed on Jun. 17, 2005, entitled “SSX-2 PEPTIDE ANALOGS”;U.S. patent application Ser. No. 11/155,929, filed on Jun. 17, 2005,entitled “NY-ESO-1 PEPTIDE ANALOGS” (Publication No. 20060094661); U.S.patent application Ser. No. 11/321,967, filed on Dec. 29, 2005, entitled“METHODS TO TRIGGER, MAINTAIN AND MANIPULATE IMMUNE RESPONSES BYTARGETED ADMINISTRATION OF BIOLOGICAL RESPONSE MODIFIERS INTO LYMPHOIDORGANS”; U.S. patent application Ser. No. 11/323,572, filed on Dec. 29,2005, entitled “METHODS TO ELICIT ENHANCE AND SUSTAIN IMMUNE REPONSESAGAINST MCH CLASS I RESTRICTED EPITOPES, FOR PROPHYLACTIC OR THERAPEUTICPURPOSES”; U.S. patent application Ser. No. 11/323,520, filed Dec. 29,2005, entitled “METHODS TO BYPASS CD4+ CELLS IN THE INDUCTION OF ANIMMUNE RESPONSE”; U.S. patent application Ser. No. 11/323,049, filedDec. 29, 2005, entitled “COMBINATION OF TUMOR-ASSOCIATED ANTIGENS INCOMPOSITIONS FOR VARIOUS TYPES OF CANCERS”; U.S. patent application Ser.No. 11,323,964, filed Dec. 29, 2005, entitled “COMBINATIONS OFTUMOR-ASSOCIATED ANTIGENS IN DIAGNOSTICS FOR VARIOUS TYPES OF CANCERS”;U.S. Provisional Application Ser. No. 60/691,889, filed on Jun. 17, 2005entitled “EPITOPE ANALOGS.”

VI. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention in designing plasmids containing immunogenic epitopesof PSMA and PRAME that are capable of eliciting a bivalent immuneresponse. It should be appreciated by those of skill in the art that themethodology disclosed in the examples which follow representmethodologies discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Design of Plasmid Expression Vectors Encoding Immunogens

The plasmids P2 and R2 (also referred to as pCTLR2) contain elementsfrom PSMA (expressed on the neovasculature of a wide range of carcinomasor by prostate carcinoma cells) and PRAME (expressed by cancerouscells), respectively, (FIG. 1). Each insert encompasses a fragment ofthe antigen's sequence along with multiple copies of an epitopeexpressed by target cells and addressable via immune mediated attack.Flanking these epitopes are sequences encoding amino acids known tofacilitate the processing and generation of epitope peptides in thecellular compartments. In addition, plasmid RP5 encompasses elementsfrom both P2 and R2 with the expressed immunogens adjoined to eachother.

The R2 plasmid was constructed following the plasmid constructionprotocol discussed above and as disclosed in U.S. Provisional PatentApplication No. 60/691,889, filed on Jun. 17, 2005 entitled EPITOPEANALOGS; and U.S. patent application Ser. No. 11/455,278 entitled PRAMEEPITOPE ANALOGS, incorporated herein by reference in its entirety. R2,also referred to as pCTLR2, is a recombinant DNA plasmid vaccine whichencodes one polypeptide with an HLA A2-specific CTL epitope from PRAME,SLLQHLIGL (SEQ ID NO:5), amino acid residues 425-433, and an epitopecluster region of PRAME, amino acids 422-509 (SEQ ID NO:21). Inconstructing this plasmid, the DNA sequence encoding the polypeptide inthe plasmid is placed under the control of promoter/enhancer sequencefrom cytomegalovirus (CMVp) which allows efficient transcription ofmessenger for the polypeptide upon uptake by antigen presenting cells. Abovine growth hormone polyadenylation signal (BGH polyA) at the 3′ endof the encoding sequence provides signal for polyadenylation of themessenger to increase its stability as well as translocation out ofnucleus into the cytoplasm. To facilitate plasmid transport into thenucleus, a nuclear import sequence (NIS) from Simian virus 40 has beeninserted in the plasmid backbone. One copy of CpG immunostimulatorymotif is engineered into the plasmid to further boost immune responses.Additionally, two prokaryotic genetic elements in the plasmid areresponsible for amplification in E. coli, the kanamycin resistance gene(Kan R) and the pMB bacterial origin of replication.

The amino acid sequence (SEQ ID NO:10) of the encoded polypeptide of R2(pCTLR2) is 150 amino acid residues in length as shown below:

malqsllqhliglsnithylypvplesyedihgtlhlerlaylharlrellcelgrpsmvwlsanpcphcgdrtfydpepilcpcfmpnkrsllqhliglgdaaysllqhliglispekeeqyiasllqhliglkrpsikrsllqhligl

Amino acid residues 2 to 89 correspond to an epitope cluster regionrepresenting PRAME₄₂₂₋₅₀₉ (SEQ ID NO:21). Within this epitope clusterregion, a number of potential HLA A2-specific CTL epitopes have beenfound using a variety of epitope prediction algorithms. Amino acidresidues 90-150 are an epitope liberation (Synchrotope™) sequence withfour embedded copies of the PRAME₄₂₅₋₄₃₃ (SEQ ID NO:5) CTL epitope(boldface). Flanking the defined PRAME CTL epitope are short amino acidsequences that have been shown to play an important role in theprocessing of the PRAME CTL epitope. In addition, the amino acidsequence ISPEKEEQYIA (SEQ ID NO: 28; corresponding to PRAME amino acid276-286, in italics) is engineered into this string-of-beads region tofacilitate the antibody-based detection of expression of encodedpolypeptide.

Example 2 Dominant/Subdominant Hierarchy of Engineered ImmunogenicElements

A study was conducted to assess whether the strategy of engineeringelements from different antigens into the same expression vector createsa dominant/subdominant hierarchy amongst those elements.

Four groups of HHD transgenic mice were immunized with plasmids P2, R2,RP5 or a mixture of P2 and R2 plasmids, by direct inoculation into theinguinal lymph nodes of 25 μg/plasmid in 25 μl of PBS to each lymph nodeat day 1, 4, 15 and 18. Ten days after the boost, splenocytes werestimulated ex vivo with PRAME₄₂₅₋₄₃₃ (SEQ ID NO:5) or PSMA₂₈₈₋₂₉₇ (SEQID NO:6) peptide and tested against ⁵¹Cr-labeled peptide coated-T2cells, at various effector to target cell ratios (E:T ratio).

Briefly, target cells expressing antigen on their surface were labeledwith a radioactive isotope of chromium (⁵¹Cr). Splenocytes were thenmixed with the target cell and incubated for several hours. Afterincubation, supernatants were harvested and the cytolytic activity wasmeasured in triplicate samples using a gamma counter. Lysis ofantigen-expressing cells releases ⁵¹Cr into the medium. Cell-specificlysis is calculated by comparing lysis (i.e., chromium release) oftarget cells expressing the antigen(s) of interest or control antigen(s)in the presence or absence of effector cells, and is usually expressedas the % specific lysis.

The corrected percent lysis was calculated for each concentration ofeffector cells, using the mean cpm for each replicate of wells (FIG. 2).Percent specific lysis was calculated using the following formula:Percent release=100×(Experimental release−spontaneous release)/(Maximumrelease−spontaneous release). Data are presented as follows: the x-axisshows the effector to target ratio; the y-axis shows the correspondingpercentage specific lysis. Results are expressed as % specificcytotoxicity (the plasmid R2 is also referred to a CTLR2).

The results show that P2 and R2 separately elicit significant cytotoxicimmune responses. However, when the immunogens of P2 and R2 areintegrated within the RP5 plasmid, immunity against the PRAME epitope ispreserved while response to PSMA₂₈₈₋₂₉₇ (SEQ ID NO:6) epitope iseclipsed. This indicates that from an immunological standpoint ahierarchy is established between these two epitopes. Admixing P2 and R2plasmids restores bivalent immunity.

Example 3 Structure of Additional Plasmids

To design expression vectors that result in a more balanced immunityagainst both PRAME and PSMA epitopes (dominant and subdominant in thecontext of RP5), a set of immunogens was designed and incorporatedwithin the same plasmid backbone by employing various combinations ofthe three following methods:

-   -   1) The ratio between the copy numbers of the PRAME₄₂₅₋₄₃₃ (SEQ        ID NO:5) (dominant) epitope and that of the PSMA₂₈₈₋₂₉₇        (subdominant) epitope was adjusted in favor of the latter.    -   2) The less dominant epitope was placed in the C terminal        position so that it would have the proper C-terminus independent        of proteasomal processing.    -   3) The less dominant epitope (PSMA) was mutated (one or multiple        copies within the expressed insert) to improve intrinsic        immunogenic properties such as binding to, and half-life on,        class I MHC.

FIG. 3 shows the design of the various plasmids made. In FIG. 3, “V”corresponds to PSMA₂₆₆₋₂₉₇ (SEQ ID NO:29) epitopes that carry an I297Vmutation.

Example 4 Induction of Bivalent Responses Achieved by PlasmidsEncompassing Epitopes from Different Antigens

The plasmids designed as described in Example 3 above, were tested todetermine their ability to prime a bivalent immune response against thePRAME₄₂₅₋₄₃₃ (SEQ ID NO:5) and PSMA₂₈₈₋₂₉₇ (SEQ ID NO:6) tumorassociated antigens.

Six groups of HHD transgenic mice (n=8/group) were immunized withplasmids (RP8, RP9, RP10, RP11, RP12, or RP13) carrying inserts depictedin FIG. 3, by direct inoculation into the inguinal lymph nodes of 25 μgin 25 μl of PBS to each lymph node on day 1 and 4. Seven days after thelast plasmid injection, on day 11, all of the immunized animals weresacrificed including five naive controls. ELISPOT analysis was conductedby measuring the frequency of IFN-γ producing spot forming colonies(SFC), as described below.

Briefly, spleens were isolated on day 11 from euthanized animals and themononuclear cells, after density centrifugation (Lympholyte Mammal,Cedarlane Labs, Burlington, N.C.), were resuspended in HL-1 medium.Splenocytes (5×10⁵ or 2.5×10⁵ cells per well) were incubated with 10 μgof PSMA₂₈₈₋₂₉₇ (SEQ ID NO:6) or PRAME₄₂₅₋₄₃₃ (SEQ ID NO:5), naturalpeptide in triplicate wells of a 96 well filter membrane plates(Multi-screen IP membrane 96-well plate, Millipore, Mass.). Samples wereincubated for 42 hours at 37° C. with 5% CO₂ and 100% humidity prior todevelopment. Mouse IFN-γ coating antibody (IFN-γ antibody pair, U-CyTechBiosciences, The Netherlands) was used as a coating reagent prior toincubation with splenocytes, followed by the accompanied biotinylateddetection antibody. GABA conjugate and proprietary substrates fromU-CyTech Biosciences were used for IFN-γ spot development. The CTLresponse in immunized animals was measured 24 hours after development onthe AID International plate reader using ELISpot Reader software version3.2.3 calibrated for IFN-γ spot analysis.

The results depicted in FIG. 4 show the average IF′N-γ spot count foreach experimental group. The data are presented as the frequency ofIF′N-γ spots (representing a colony of IFN-γ secreting cells) pertreatment, as the average of individual animal responses +/− thestandard deviation (Std). Data generated from splenocytes isolated fromimmunized or naive mice and stimulated with the PSMA₂₈₈₋₂₉₇ nativepeptide indicated that RP12 induced the strongest immunity to thePSMA₂₈₈₋₂₉₇ antigen (55.8 +/−1.6 INF-γ spots) as compared to the naivecontrol (12.8+2.4 IFN-γ spots) or the other treatment groups. Thisrepresented a 5-fold enhanced PSMA immune response with the RP12plasmid.

In addition, data generated from splenocytes isolated from immunized ornaive mice and stimulated with the PRAME₄₂₅₋₄₃₃ native peptide alsodemonstrated that animals immunized with RP12 showed the strongestimmune response to the PRAME₄₂₅₋₄₃₃ antigen (234.5+3.7 IFN-γ spots) ascompared to the naive controls (8.5+2.8 IFN-γ spots) or the othertreatment groups. This represented a greater than 20-fold increasedPRAME response with the RP12 plasmid.

Overall, the results depicted in FIG. 4 show induction of a strongbivalent immunity against both PRAME and PSMA epitopes by the plasmidRP12. Some bivalent immunity against both PRAME and PSMA epitopes wasobserved with RP9 and to a lesser extent RP13, RP10, RP11 and RP8—allhaving a more potent representation of the PSMA epitope relative toPRAME epitope as compared to the plasmid RP5 (FIG. 2). This observationis apparently due in part to use of the I297V analogue of PSMA.

Example 5 Induction of a Bivalent Response by the RP12 Plasmid

Based on the comparison in Example of 4 of the six plasmids (RP8, RP9,RP10, RP11, RP12, and RP13), RP12 was selected for further analysis, asit was the only plasmid that primed a robust, bivalent immune responseagainst both PRAME₄₂₅₋₄₃₃ (SEQ ID NO:5) and PSMA₂₈₈₋₂₉₇ (SEQ ID NO:6).

Two representative HHD transgenic mice were immunized with RP12 plasmidcarrying an insert (depicted in FIG. 3) by direct inoculation into theinguinal lymph nodes of 25 μg in 25 μl of PBS to each lymph node at day1, 4, 15 and 18. Ten days after the last plasmid injection, thefrequency of PRAME and PSMA epitope-specific CD8⁺ T cells was measuredby tetramer staining of peripheral blood mononuclear cells andco-staining for CD8 expression.

Briefly, mononuclear cells were isolated from peripheral blood afterdensity centrifugation (Lympholyte Mammal, Cedarlane Labs) and stainedwith HLA-A*0201 PRAME MHC tetramer (Beckman Coulter, T02001), andHLA-A*0201 PSMA MHC tetramer (Beckman Coulter, T02001). These cells werethen co-stained using FITC conjugated rat anti-mouse CD8a (Ly-2)monoclonal antibody (BD Biosciences, 553031). Data were collected usinga BD FACS Calibur flow cytometer and analyzed using Cellquest softwareby gating on the lymphocyte population and calculating the percent oftetramer positive cells within the CD8′ CTL population.

The results depicted in FIG. 5, show that following intranodal plasmidimmunization, the RP12 plasmid elicited dual immunity and the frequencyof PRAME₄₂₅₋₄₃₃ specific T cells was several fold higher than that of Tcells specific for the PSMA subdominant epitope

Example 6 Bivalent Immune Response in Mice Primed with PRAME and PSMAPlasmid and Boosted with Peptide

To determine whether immunization with the plasmids RP12 and RP8 couldinduce a bivalent response against the tumor associated antigensPrame₄₂₅₋₄₃₃ and PSMA₂₈₈₋₂₉₇, following peptide boost with thePSMA₂₈₈₋₂₉₇ I297V (SEQ ID NO:7) analogue, a tetramer analysis ofimmunized animals was conducted.

HHD transgenic mice were immunized with RP8 or RP12 plasmids carryinginserts depicted in FIG. 3, by direct inoculation into the inguinallymph nodes of 100 μg in 25 μl of PBS to each lymph node at day 1, 4, 15and 18. On days 29 and 32, the mice were boosted with 25 μg ofPSMA₂₈₈₋₂₉₇ peptide analogue (I297V). One day before initiation ofpeptide boost and ten days after the completion of plasmid boost, thefrequency of PRAME and PSMA epitope-specific T cells was measured bytetramer staining (as described above) and compared to tetramer resultsseven days following the last peptide boost.

The results shown in FIG. 6, as mean±SEM of specific CD8⁺T cellfrequency showed that RP12 plasmid elicits a slightly higherPSMA-specific immunity than RP8 prior to peptide boost. In addition, inboth the case of RP12 and RP8, the immunity against PRAME was found tobe dominant prior to peptide boost. However, after boost with the PSMAsubdominant epitope, the immune response against PRAME and PSMAdisplayed a more balanced profile, particularly in the case of RP12,indicating the benefit of strategies to elicit equilibrated immuneresponses against epitopes of different immune hierarchy.

FIG. 6B shows immune responses to PRAME and PSMA in three representativemice from each group and further illustrate the enhanced bivalentresponse elicited by the RP12 plasmid and the PSMA (I297V) peptideboost.

Example 7 Bivalent Immune Response after PSMA Peptide Boost andSubsequent PRAME Peptide Boost

It was examined whether immunization with the plasmids RP12 and RP8could induce a bivalent response against the PRAME₄₂₅₋₄₃₃ (SEQ ID NO:5)and PSMA₂₈₈₋₂₉₇ (SEQ ID NO:6) epitopes those tumor associated antigens,following a first peptide boost with the PSMA₂₈₈₋₂₉₇ I297V (SEQ ID NO:7)analogue and a second boosting with PRAME₄₂₅₋₄₃₃ L426Nva, L433Nle (SEQID NO:30) peptide analogue.

Mice were immunized with 4 injections of the RP8 or RP12 plasmid (4mg/ml) by direct inoculation into the inguinal lymph nodes at day 1, 4,15 and 18. On days 29 and 32, the mice were boosted with PSMA₂₈₈₋₂₉₇I297V peptide analogue (0.5 mg/ml), followed by a second boost on day 42and 59 with Prame₄₂₅₋₄₃₃ L426Nva, L433Nle peptide analogue at 0.5 mg/mland 0.05 mg/ml respectively. Mice were sacrificed and an ELISPOTanalysis (FIG. 7) was conducted as follows.

Briefly, spleens were isolated ten days following the last Prame₄₂₅₋₄₃₃L426Nva, L433Nle peptide injection from euthanized animals and themononuclear cells, after density centrifugation (Lympholyte Mammal,Cedarlane Labs, Burlington, N.C.), were resuspended in HL-1 medium.Splenocytes (2×10⁵ cells per well) were incubated with 10 μg ofPSMA₂₈₈₋₂₉₇ or PRAME₄₂₅₋₄₃₃, natural peptide in triplicate wells of a 96well filter membrane plates (Multi-screen IP membrane 96-well plate,Millipore, Mass.). Samples were incubated for 72 hours at 37° C. with 5%CO₂ and 100% humidity prior to development. Mouse IFN-γ coating antibody(IFN-γ antibody pair, U-CyTech Biosciences, The Netherlands) was used asa coating reagent prior to incubation with splenocytes, followed by theaccompanied biotinylated detection antibody. GABA conjugate and varioussubstrates from U-CyTech Biosciences were used for IFN-γ spotdevelopment. The CTL response in immunized animals was measured 24 hoursafter development on the AID International plate reader using ELISpotReader software version 3.2.3 calibrated for IFN-γ spot analysis.

The results show that RP12 plasmid elicits a higher PSMA-specificimmunity than RP8 at all doses tested. For both the RP12 and RP8plasmids, the immunity against PRAME was found to be dominant at alldoses tested. Also, the RP12 plasmid showed a strong balanced immuneresponse against PRAME and PSMA following boost with the PSMA and PRAMEepitopes.

1. An engineered nucleic acid construct encoding a polypeptidecomprising an array of epitopes, wherein the array comprises one or morecopies of CTL epitope PSMA₂₈₈₋₂₉₇ (SEQ ID NO:6), or a cross-reactiveanalogue differing from SEQ ID NO:6 by only 1-3 substitutions, and oneor more copies of CTL epitope PRAME₄₂₅₋₄₃₃ (SEQ ID NO:5), or across-reactive analogue differing from SEQ ID NO:5 by only 1-3substitutions, wherein the polypeptide does not comprise a whole PSMAantigen (SEQ ID NO: 19), and wherein the polypeptide does not comprise awhole PRAME antigen (SEQ ID NO: 20).
 2. The nucleic acid construct ofclaim 1, wherein one or both epitopes are encoded within a liberationsequence.
 3. The nucleic acid construct of claim 1, wherein saidpolypeptide further comprises a sequence encoding one or more epitopeclusters.
 4. The nucleic acid construct of claim 3 comprising a PRAMEepitope cluster.
 5. The nucleic acid construct of claim 4, wherein saidepitope cluster consists essentially of amino acids 422-509 of PRAME(SEQ ID NO:21).
 6. The nucleic acid construct of claim 3 comprising aPSMA epitope cluster.
 7. The nucleic acid construct of claim 6, whereinsaid epitope cluster is chosen from the group consisting of amino acids3-45 of PSMA (SEQ ID NO:22) and 217-297 of PSMA (SEQ ID NO:23).
 8. Thenucleic acid construct of claim 1, wherein the cross-reactive analogueof CTL epitope PSMA₂₈₈₋₂₉₇ (SEQ ID NO:6) comprises a I297V substitution.9. The nucleic acid construct of claim 1 further comprising a nuclearimport sequence.
 10. The nucleic acid construct of claim 1 furthercomprising one or more of a CpG immunostimulatory motif.
 11. The nucleicacid construct of claim 2, wherein the liberation sequence of the PRAMEand PSMA epitope is located in the N-terminal portion of the encodedpolypeptide.
 12. The nucleic acid construct of claim 11, wherein theencoded polypeptide is SEQ ID NO:2.
 13. The nucleic acid construct ofclaim 2, wherein the liberation sequence of the PRAME and PSMA epitopesis located in the C-terminal portion of the encoded polypeptide.
 14. Thenucleic acid construct of claim 13, wherein the encoded polypeptide isSEQ ID NO:4.
 15. An immunogenic composition comprising the nucleic acidconstruct of claim
 1. 16. The nucleic acid construct of claim 2, whereinboth epitopes are encoded within a liberation sequence.
 17. The nucleicacid construct of claim 1, wherein the array comprises a flanking aminoacid sequence adjacent to a PSMA₂₈₈₋₂₉₇ epitope for liberation of theepitope by immunoproteasome processing.
 18. The nucleic acid constructof claim 1, wherein the array comprises a flanking amino acid sequenceadjacent to a PRAME₄₂₅₋₄₃₃ epitope for liberation of the epitope byimmunoproteasome processing.
 19. The nucleic acid construct of claim 17,wherein the flanking amino acid sequence is not an epitope.
 20. Thenucleic acid construct of claim 18, wherein the flanking amino acidsequence is not an epitope.
 21. The nucleic acid construct of claim 19,wherein the flanking amino acid sequence is at least 2 amino acids inlength.
 22. The nucleic acid construct of claim 20, wherein the flankingamino acid sequence is at least 2 amino acids in length.